The intracellular delivery of biologically active agents, for example, pharmacologically active materials and diagnostic agents, is generally desirable in connection with the treatment and/or diagnosis of various diseases. For example, cell function can be influenced at the subcellular or molecular level by delivering the biologically active agent intracellularly.
Various methods have been developed for the delivery of biologically active agents directly into living cells. Included among such methods is the "carrier method" which involves the use of a carrier to promote intracellular delivery of a bioactive agent to specifically targeted cells, for example, diseased cells. The intracellular delivery of therapeutic agents is referred to herein also as "transfection".
Various carriers have been developed for use in the transfection of biologically active agents. For example, liposomes and polymers have been developed for the transfection of genetic materials, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). However, the currently available carriers, including liposomes and polymers, are generally ineffective for the intracellular delivery of biologically active materials in vivo. Moreover, the currently available carriers have limited use in connection with the transfection of cells in vitro.
In addition to the carrier method, alternative methods have been developed for the transfection of biologically active agents, including genetic material, directly into cells. These methods include, for example, calcium phosphate precipitation and electroporation. However, these methods are also generally ineffective for the intracellular delivery of biologically active agents in vivo.
Great strides have been made in connection with the characterization and understanding of various diseases, for example, genetic diseases, and their associated protein transcription, in humans and other animals. This has led to the development or postulation of improved methods for the treatment of such diseases with biologically active agents. Various of these methods involve or require that the biologically active agent be delivered intracellularly. As noted above, however, current methods for the transfection of cells with biologically active agents in vivo are generally ineffective. This is thwarting the study and implementation of improved methods for the treatment of various diseases.
The cellular membrane is a selective barrier which prevents random introduction of substances into the cell. Accordingly, a major difficulty in the intracellular delivery of biologically active agents is believed to involve the transfer of the agent from the extracellular space to the intracellular space. Localization of the biologically active agent at the surface of selected cell membranes has been difficult to achieve also.
Carriers have been engineered also from viral vectors. Specifically, vectors for the transfection of genetic material have been developed from whole viruses, including adenoviruses and retroviruses. However, only a limited amount of biologically active materials can be placed inside of a viral capsule. Moreover, in the case of biologically active materials which comprise genetic material, undesired interaction of the viral carrier may occur with the encapsulated genetic material and the patient.
To minimize the potential interactions associated with viruses, attempts have been made to use only certain components of a virus. This is difficult to achieve in vivo inasmuch as the virus components must be able to recognize and reach the targeted cells. Despite extensive work, a successfully targeted, viral-mediated vector for the delivery of biologically active materials into cells in vivo has not been adequately achieved.
As noted above, liposomes have been used as a carrier for the intracellular delivery of biologically active agents, including genetic material. One of the original methods for the use of liposomes as carriers for biologically active agents is disclosed in Szoka and Papahadjopoulos, Ann. Rev. Biophysic. Bioeng., Vol. 9, pp. 467-508 (1980). The disclosed method involves the preparation of liposomes by the addition of an aqueous solution of genetic material to phospholipids which are dissolved in ether. Evaporation of the ether phase provides genetic material encapsulated in lipid vesicles.
Another method for encapsulating biologically active agents in liposomes involves the extrusion of dehydration-rehydration vesicles. Other methods, in addition to those described above, are known for the encapsulation by liposomes of biologically active agents.
More recently, liposomes have been developed from cationic lipids, such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride ("DOTMA") or lipids which comprise cationic polymers, for example, polysine. See, e.g., Xiaohuai and Huang, Biochimica et Biophysica Acta, Vol. 1189, pp. 195-203 (1994). Liposomes which are prepared from cationic materials (referred to hereinafter as "cationic liposomes") have been developed, inter alia, to transfect cells with genetic material, including DNA. It is believed that the cationic liposomes bind with the negatively charged phosphate group(s) of the nucleotides in DNA. Studies have shown that cationic liposomes mediate transfection of cells with genetic material in vitro more efficiently than other carriers, for example, cationic polymers. In addition, in vitro studies have shown also that cationic liposomes provide improved transfection of cells relative to other delivery methods, including electroporation and calcium phosphate precipitation.
However, the currently available cationic lipids and cationic liposomes are generally ineffective for the intracellular delivery of biologically active agents in vivo. Moreover, they are generally ineffective for the intracellular delivery of biologically active agents in serum. This is a serious drawback inasmuch as cells require serum for viability. In fact, it is generally necessary to remove serum from tissue culture baths during gene transfection studies involving cationic lipids and cationic liposomes. After transfection, the serum is replaced. This involves additional processing steps which render transfection of cells with cationic lipids and cationic liposomes complex and cumbersome.
New and/or better cationic lipids useful, inter alia, for the intracellular delivery of bioactive agents are needed. The present invention is directed to this as well as other important ends.